Method for molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase

ABSTRACT

This invention provides a modified vaccinia topoisomerase enzyme containing an affinity tag which is capable of facilitating purification of protein-DNA complexes away from unbound DNA. This invention further provides a modified sequence specific topoisomerase enzyme. 
     This invention provides a method of ligating duplex DNAs, a method of molecular cloning of DNA, a method of synthesizing polynucleotides, and a method of gene targeting. 
     Lastly, this invention provides a recombinant DNA molecule composed of segments of DNA which have been joined ex vivo by the use of a sequence specific topoisomerase and which has the capacity to transform a suitable host cell comprising a DNA sequence encoding polypeptide activity.

This application is a continuation of U.S. Ser. No. 11/368,299, filedMar. 3, 2006 now U.S. Pat. No. 7,550,295, which is a continuation ofU.S. Ser. No. 10/360,478, filed Feb. 7, 2003, now U.S. Pat. No.7,026,141, issued Apr. 11, 2006, which is a continuation of U.S. Ser.No. 08/898,517, filed Jul. 22, 1997, now U.S. Pat. No. 6,548,277, issuedApr. 15, 2003, which is a divisional of U.S. Ser. No. 08/358,344, filedDec. 19, 1994, now U.S. Pat. No. 5,766,891, issued Jun. 16, 1997, thecontents of all of which are hereby incorporated by reference in theirentirety into the present application.

This invention was made with support under Grant No. GM-46330 from theNational Institutes of Health, U.S. Department of Health and HumanServices. Accordingly, the United States Government has certain rightsin the invention.

Throughout this application, various publications are referenced byArabic numerals in brackets. Full citations for these publications maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications are in their entiretyhereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Construction of chimaeric DNA molecules in vitro relies traditionally ontwo enzymatic steps catalyzed by separate protein components.Site-specific restriction endonucleases are used to generate linear DNAswith defined termini that can then be joined covalently at their endsvia the action of DNA ligase.

Vaccinia DNA topoisomerase, a 314-aa virus-encoded eukaryotic type Itopoisomerase [11], binds to duplex DNA and cleaves the phosphodiesterbackbone of one strand. The enzyme exhibits a high level of sequencespecificity, akin to that of a restriction endonuclease. Cleavage occursat a consensus pentapyrimidine element 5′-(C/T)CCTT^(Ø) (SEQ ID NO:16)in the scissile strand [12, 5, 6]. In the cleavage reaction, bond energyis conserved via the formation of a covalent adduct between the 3′phosphate of the incised strand and a tyrosyl residue (Tyr-274) of theprotein [10]. Vaccinia topoisomerase can religate the covalently heldstrand across the same bond originally cleaved (as occurs during DNArelaxation) or it can religate to a heterologous acceptor DNA andthereby create a recombinant molecule [7, 8].

The repertoire of DNA joining reactions catalyzed by vacciniatopoisomerase has been studied using synthetic duplex DNA substratescontaining a single CCCTT (SEQ ID NO:17) cleavage site. When thesubstrate is configured such that the scissile bond is situated near(within 10 bp of) the 3′ end of a DNA duplex, cleavage is accompanied byspontaneous dissociation of the downstream portion of the cleaved strand[4]. The resulting topoisomerase-DNA complex, containing a 5′single-stranded tail, can religate to an acceptor DNA if the acceptormolecule has a 5′ OH tail complementary to that of the activated donorcomplex. Sticky-end ligation by vaccinia topoisomerase has beendemonstrated using plasmid DNA acceptors with four base overhangscreated by restriction endonuclease digestion [8].

SUMMARY OF THE INVENTION

This invention provides a modified vaccinia topoisomerase enzymecontaining an affinity tag which is capable of facilitating purificationof protein-DNA complexes away from unbound DNA. This invention furtherprovides a modified sequence specific topoisomerase enzyme.

This invention provides a method of ligating duplex DNAs, a method ofmolecular cloning of DNA, a method of synthesizing polynucleotides, anda method of gene targeting.

Lastly, this invention provides a recombinant DNA molecule composed ofsegments of DNA which have been joined ex vivo by the use of a sequencespecific topoisomerase and which has the capacity to transform asuitable host cell comprising a DNA sequence encoding polypeptideactivity.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C: Sticky-End Ligation.

FIG. 1A: Topoisomerase-mediated cleavage of a 24-nucleotideCCCTT-containing hairpin substrate (SEQ ID NOs:6 and 7) was assayed as afunction of enzyme concentration.

The structure of the substrate is shown; the site of strand scission isindicated by the arrow. Reaction mixtures (20 ml) containing 50 mM TrisHCl (pH 7.5), 0.5 pmol of 5′ ³²P-labeled DNA, and topoisomerase wereincubated at 37° C. for 5 min. Covalent complexes were trapped byaddition of SDS to 1%. Samples were then electrophoresed through a 10%polyacrylamide gel containing 0.1% SDS. Covalent complex formation wasrevealed by transfer of radiolabeled DNA to the topoisomerasepolypeptide as detected by autoradiographic exposure of the dried gel.The extent of adduct formation was quantitated by scintillation countingof an excised gel slice containing the labeled protein and was expressedas the percent of the input 5′ ³²P-labeled oligonucleotide that wascovalently transferred to protein.

FIG. 1B: Reaction mixtures containing 50 mM Tris HCl (pH 7.5), 460 fmolof 5′ ³²P-labeled hairpin substrate, and 2 pmol of topoisomerase wereincubated for 5 min at 37° C., then supplemented with linear pUC18 DNAacceptor (350 fmol of ends) as indicated and incubated for another 5 minat room temperature. Samples were adjusted to 0.2 M NaCl and 0.5% SDS,then electrophoresed through a 1.2% agarose gel in TBE (90 mM Tris, 90mM borate, 2.5 mM EDTA) with 0.5 mg/ml ethidium bromide. DNA wasvisualized by photographing the stained gel under short wave UVillumination.

FIG. 1C: The same gel was then dried and exposed for autoradiography.The positions of the radiolabeled topoisomerase-DNA “donor” complex andthe pUC strand transfer product are indicated at the right. pUC18 DNAused as acceptor in the strand transfer reactions was linearizedquantitatively by digestion with a single-cut restriction enzyme. The 5′phosphate termini of the linear DNAs were converted to 5′ OH ends bytreatment of the DNAs with calf intestinal phosphatase as indicated(CIP). The acceptor DNAs included in each reaction are specifiedaccording to lane number. Lane M (left panel) contains DNA size markers(l HindIII digest).

FIG. 2: Monovalent, Bivalent, and Trivalent Substrates.

The structure of the complementary hairpin oligonucleotides S300 (SEQ IDNO:8) and S301 (SEQ ID NO:9) are shown. The 5′ terminus is indicated byan asterisk. The CCCTT recognition site of topoisomerase cleavage isunderlined. The structure of the bivalent linker DNA formed by annealingS300 and S301 strands is shown in the middle. At bottom is the structureof the trivalent Y-branched linker formed by annealing S300, S304, andS303 oligonucleotides.

FIGS. 3A-3C: Topoisomerase-Mediated Cleavage of Monovalent, Bivalent,and Trivalent Substrates.

FIG. 3A: Radiolabeled cleavage substrates were electrophoresed through anative polyacrylamide gel (7.5% acrylamide, 0.2% bisacrylamide) in TBEat 100 V. An autoradiogram of the dried gel is shown. Lane 1 containsthe 5′ ³²P-46-mer “flip” hairpin (S300 (SEQ ID NO:8); FIG. 2). Lane 2contains the 46-bp divalent cleavage substrate (FIG. 2). This structurewas formed by annealing the 5′ ³²P-S300 strand to a 3-fold molar excessof unlabeled 46-nt complementary strand (S301 (SEQ ID NO:9), or “flop”strand; FIG. 2). Lane 3 contains the trivalent Y-branch substrate formedby annealing 5′ ³²P-S300 to two unlabeled 46-mer oligos (S303 and S304),each present at 3-fold molar excess over the labeled strand.

FIG. 3B: Cleavage reaction mixtures (20 ml) contained 50 mM Tris HCl (pH7.5), 0.6 pmol of 5′ ³²P-labeled DNA, and 20 pmol of topoisomerase(lanes 2, 4, 6, and 8) were incubated at 37° C. for 5 min. Enzyme wasomitted from control reactions (lanes 1, 3, 5, and 7). Covalentcomplexes were trapped by addition of SDS to 1%. (Note that the sampleswere not heat-denatured). Labeled cleavage products were resolved bySDS-PAGE. Free DNA migrated with the bromophenol blue dye front. Thestructures of the various covalent protein-DNA complexes are indicatedat the right of the autoradiogram. The positions and sizes (in kDa) ofprestained marker proteins are indicated at the left. The inputsubstrates are illustrated at the bottom of the autoradiogram: *S300(lanes 1 and 2); *S301 (lanes 3 and 4); *S300/S301 (lanes 5 and 6);S300/*S301 (lanes 7 and 8).

FIG. 3C: Cleavage reactions contained 0.36 pmol of radiolabeled Y-branchsubstrate (*S300/S303/S304) and 20 pmol of topoisomerase (lane 2).Enzyme was omitted from a control reaction (lane 1). The structures ofthe various covalent protein-DNA complexes are indicated at the right ofthe autoradiogram. The positions and sizes (in kDa) of prestained markerproteins are indicated at the left.

FIGS. 4A-4B: Topoisomerase-Mediated Joining of Two Ends Via a BivalentLinker.

FIG. 4A: Reaction mixtures (20 ml) contained 50 mM Tris HCl (pH 7.5), 2pmol of topoisomerase, and either 5′ ³²P-labeled monovalent substrate(*S300, 0.6 pmol—lanes 1 and 2) or 5′ ³²P-labeled bivalent linker (0.3pmol of *S300/S301, i.e., 0.6 pmol of ends—lanes 3 and 4). Afterincubation for 5 min at 37° C., the reactions were supplemented with5′-OH HindIII-cut pUC18 DNA acceptor (380 fmol of ends) as indicated andincubated for another 5 min at room temperature. Samples were adjustedto 0.2 M NaCl and 0.5% SDS, then electrophoresed through a 1.2% agarosegel in TBE. The ethidium bromide stained gel is shown at left. Thepositions and sizes (kbp) of marker DNA fragments (lane M) are indicatedat the left.

FIG. 4B: The same gel was dried and exposed for autoradiography. Thepositions of the radiolabeled topoisomerase-DNA “donor” complex and thestrand transfer products are indicated at right by arrows.

FIGS. 5A-5D: Molecular Cloning of DNA Using Vaccinia Topoisomerase.

FIG. 5A: Ligation reactions for topoisomerase-based cloning wereperformed as described under Experimental Details. The protocol isillustrated schematically.

FIGS. 5B-5C: Plasmid DNA was prepared from bacteria containing pUC18(the parent vector, FIG. 5B) and pUC-T11 (a representative transformantfrom the topoisomerase ligation reaction, FIG. 5C). DNA was digestedwith the restriction endonucleases specified above each lane usingreaction buffers provided by the vendor. Undigested plasmid DNA is shownin Lane “--”. Lane M contains DNA size markers. The positions and sizes(kbp) of reference fragments are indicated.

FIG. 5D: The structure of the 46-bp bivalent linker (SEQ ID NOs:10 and11) is indicated. Diagnostic restriction sites within the linker arespecified above the sequence.

FIGS. 6A-6B: Topoisomerase-Mediated Joining of Two Ends Via a TrivalentLinker.

FIG. 6A: Each strand of the trivalent substrate (FIG. 2) was 5′ labeledand gel-purified. The Y-branched substrate was generated by annealingequimolar amounts of the three strands (*S300, *S303, *S304). Theannealed product was analyzed by electrophoresis through a native 7.5%polyacrylamide gel. An autoradiograph of the gel is shown. The trivalentsubstrate is in lane 3. Component strands were analyzed in parallel(*S303 in lane 1; *S304 in lane 2). The structures of the labeledspecies are indicated at the right.

FIG. 6B: Reaction mixtures (20 ml) contained 50 mM Tris HCl (pH 7.5), 1pmol of topoisomerase, and either 5′ ³²P-labeled monovalent substrate(*S304—lanes 1 and 2) or 5′ ³²P-labeled trivalent linker (0.3 pmol of*S300/*S303/*S304—lanes 3 and 4). Each reaction contained 350 fmol ofinput substrate (expressed as cleavable ends). After incubation for 5min at 37° C., the reactions were supplemented with 5′-OH HindIII-cutpUC18 DNA acceptor (570 fmol of ends) as indicated and incubated foranother 5 min at room temperature. Samples were adjusted to 0.2 M NaCland 0.5% SDS, then electrophoresed through a 1.2% agarose gel in TBE.The ethidium bromide stained gel is shown. The positions and sizes (kbp)of marker DNA fragments (lane M) are indicated at the left.

FIG. 6C: The same gel was dried and exposed for autoradiography. Thepositions of the radiolabeled topoisomerase-DNA “donor” complex and thestrand transfer products are indicated at right by arrows and brackets.

FIG. 7: Expected Products of Bivalent End-Joining.

The locations of restriction sites for HindIII (H), XmnI (X), SspI (S),and AccI (A) within the linear pUC acceptors and anticipated ligationproducts are indicated by arrows. The pUC DNA is denoted by a solid bar.The predicted sizes of SspI and XmnI restriction fragments derived fromeach species are listed at the left. Fragments that are expected tocontain radiolabeled linker DNA are indicated by asterisks.

FIG. 8: Expected Products of Trivalent End-Joining.

The expected products of trivalent end joining to pUC DNA are shown inthe box. Digestion with XmnI is predicted to yield four trivalentproducts, which are depicted at the right. The lengths of the pUC “arms”(in kpb) are indicated.

FIGS. 9A-9C: Restriction Endonuclease Digestion of End-Joining ReactionProducts.

FIG. 9A: Reaction mixtures (20 ml) contained 50 Mm Tris Hcl (pH 7.5), 1pmol of topoisomerase, and either monovalent substrate (*S300—lanes 1and 2), divalent linker (*S300/*301—lanes 3 an 4), or trivalent linker(*S300/*S303/*S304—lanes 5 and 6). After incubation for 5 min at 37° C.,the reactions were supplemented with either 5′-OH HindIII-cut pUC19“bivalent” DNA acceptor (600 fmol linear DNA—lanes 1, 3, and 5) or 5′-OHHindIII/5′-P AccI-cut PUC19 “monovalent” acceptor (500 fmol of linearDNA—lanes 2, 4, and 6) and incubated for another 5 min at roomtemperature. The mixtures were adjusted to recommended restrictionconditions by addition of 10× buffer concentrate (NEB2) and the sampleswere digested with SspI (10 units; New England BioLabs) for 60 min at37° C. Samples were adjusted to 0.5% SDS and electrophoresed through a1.2% agarose gel in TBE. An ethidium bromide stained gel is shown. Thepositions and sizes (kbp) of marker DNA fragments (lane M) are indicatedat the left.

FIGS. 9B-9C: Cleavage reactions containing radiolabeled bivalent linker(lanes 1 and 2) or trivalent linker (lanes 3-5) were supplemented withdivalent pUC19 acceptor (lanes 1 and 3) or monovalent pUC19 acceptor(lanes 2 and 4). A control reaction received no acceptor (lane 5). Thestrand transfer reaction products were digested with XmnI (40 units) for2 h at 37° C., then analyzed by agarose gel electrophoresis. Theethidium bromide stained gel is shown (FIG. 9B). The positions and sizes(kbp) of marker DNA fragments (lane M) are indicated at the left of thephotograph. The same gel was dried and exposed for autoradiography (FIG.9C). The positions of the radiolabeled topoisomerase-DNA “donor” complexand the strand transfer products are indicated at right by arrows andbrackets.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a modified vaccinia topoisomerase enzymecontaining an affinity tag. The modified vaccinia topoisomerase enzymeis capable of facilitating purification of a vaccinia topoisomerase-DNAcomplex from unbound DNA. This invention also provides a modifiedsequence specific topoisomerase enzyme. The sequence specifictopoisomerase enzyme can be any site specific type I topoisomerase.

Topoisomerases are a class of enzymes that modify the topological stateof DNA via the breakage and rejoining of DNA strands. Vacciniatopoisomerase enzyme is a vaccinia virus-encoded eukaryotic type Itopoisomerase. In one embodiment vaccinia topoisomerase enzyme is a 314aa virus encoded type I topoisomerase.

In another embodiment the modified vaccinia enzyme is a site-specifictype I topoisomerase. Site-specific type I topoisomerases include, butare not limited to, viral topoisomerases such as pox virustopoisomerases. Examples of pox virus topoisomerases include shopefibroma virus and ORF virus. Other site specific topoisomerases areknown to those skilled in the art.

In another embodiment the affinity tag includes, but is not limited to,the following: a glutathione-S-transferase fusion tag, a maltose bindingprotein tag, a histidine or poly-histidine tag.

In one embodiment the vaccinia topoisomerase-DNA complex is purifiedfrom unbound DNA by binding the histidine tagged topoisomerase-DNAcomplex to a nickel column and eluting the substrate with imidazole.

This invention provides a duplex DNA molecule, that is, adouble-stranded DNA molecule, having at each end thereof the modifiedvaccinia topoisomerase enzyme.

Vaccinia topoisomerase binds to duplex DNA and cleaves thephosphodiester backbone of one strand while exhibiting a high level ofsequence specificity, cleaving at a consensus pentapyrimidine element5′-(C/T)CCTT_(↓) (SEQ ID NO:16), or related sequences, in the scissilestrand. In one embodiment the scissile bond is situated in the range of2-12 bp from the 3′ end of a duplex DNA. In another embodiment cleavablecomplex formation by vaccinia topoisomerase requires six duplexnucleotides upstream and two nucleotides downstream of the cleavagesite. Examples of vaccinia topoisomerase cleavable sequences include,but are not limited to, +6/−6 duplex GCCCTTATTCCC (SEQ ID NO:1), +8/−4duplex TCGCCCTTATTC (SEQ ID NO:2), +10/−2 duplex TGTCGCCCTTAT (SEQ IDNO:3), and +10/−2 duplex GTGTCGCCCTTA (SEQ ID NO:4).

As used herein, the term donor signifies a duplex DNA which contains aCCCTT (SEQ ID NO:17) cleavage site within 10 bp of the 3′ end and theterm acceptor signifies a duplex DNA which contains a 5′-OH terminus.Once covalently activated by topoisomerase the donor will only betransferred to those acceptor ends to which it can base pair.

This invention provides a method of ligating duplex DNAs employing themodified tagged vaccinia topoisomerase. In this method of ligation thedonor duplex DNA substrate is a bivalent donor duplex DNA substrate,that is, it contains two topoisomerase cleavage sites. One embodimentcomprises cleaving a donor duplex DNA substrate containing sequencespecific topoisomerase cleavage sites by incubating the donor duplex DNAsubstrate with a sequence specific topoisomerase to form atopoisomerase-bound donor duplex DNA strand and incubating thetopoisomerase-bound donor duplex DNA strand with a 5′hydroxyl-terminated compatible acceptor DNA, resulting in the ligationof the topoisomerase-bound donor duplex DNA strand to the DNA acceptorstrand.

Methods of cleaving DNA by incubation with enzymes and methods ofligating DNA by incubation are known to those skilled in the art. In oneembodiment the sequence specific topoisomerase is a vacciniatopoisomerase enzyme. In another embodiment the sequence specifictopoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT_(↓) (SEQ IDNO:16).

In one embodiment the desired subpopulation of DNA ligation product ispurified by introducing to the 5′ end of the donor duplex DNA anaffinity label. In a preferred embodiment the affinity label is a biotinmoiety and purification is performed by binding the biotin-ligatedproduct to streptavidin. Other purification methods are known to thoseskilled in the art.

Bivalent end-joining allows the assembly of linear concatamers frompolynucleotides with compatible ends. When the linker is designed togenerate the same overhang at each cleavage site, the strand transferproducts are randomly oriented as head-to-head, head-to tail, andtail-to-tail isomers. Control of the reaction can be easily achieved byusing a bivalent linker containing different overhangs at each cleavagesite; in this way, DNA acceptors prepared with two different restrictionenzymes can be assembled in a strictly head-to-tail fashion. Theligation can be made exclusively head-to-head by combining a symmetricbivalent linker with an acceptor DNA containing asymmetric ends.

Bivalent strand transfer also results in circularization of theacceptor, a property that can be exploited for molecular cloning. Forexample, by placing the topoisomerase cleavage sites on the insert (asynthetic bivalent substrate) and cloning the cleaved DNA into a plasmidvector. This strategy is well-suited to the cloning of DNA fragmentsamplified by PCR. To clone PCR products using vaccinia topoisomerase, itis necessary to include a 10-nucleotide sequence -5′-XXXXAAGGGC- (SEQ IDNO:5) at the 5′ end of the two primers used for amplification. The5′-XXXX segment can correspond to any 4-base overhang that is compatiblewith the restriction site into which the PCR product will ultimately becloned. The amplification procedure will generate duplex moleculescontaining the sequence -GCCCTT^(Ø)xxxx-3′ (SEQ ID NO:12) at both 3′ends (where xxxx is the complement of XXXX). Incubation of the PCRproduct with topoisomerase will result in cleavage at both termini andallow the covalently activated PCR fragment to be ligated to vector DNA,essentially as described in FIG. 5A.

This invention also provides a method of molecular cloning of DNA. Oneembodiment comprises introducing to a donor duplex DNA substrate asequence specific topoisomerase cleavage site by PCR amplifying thedonor duplex DNA molecule with oligonucleotide primers containing thesequence specific topoisomerase cleavage site; incubating the donorduplex DNA with a sequence specific topoisomerase, resulting in theformation of a sequence specific topoisomerase-donor duplex DNA complex;incubating the sequence specific topoisomerase-donor duplex DNA complexwith a plasmid vector with a 5′ overhang compatible to the donor;incubating the sequence specific topoisomerase-donor duplex DNA complexwith the plasmid vector; and transforming the plasmid vector that hasbeen incubated into a host cell.

In one embodiment the sequence specific topoisomerase is a vacciniatopoisomerase enzyme. In another embodiment the sequence specifictopoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT_(↓) (SEQ IDNO:16).

PCR amplification methods are known to those skilled in the art. In oneembodiment, the cloning of PCR products using vaccinia topoisomeraserequires including a 10-nucleotide sequence 5′-XXXXAAGGGC- (SEQ ID NO:5)at the 5′ end of the two primers used for amplification. The 5′-XXXXsegment can correspond to any 4-base overhang compatible with therestriction site into which the PCR product will be cloned. Theamplification procedure will generate duplex molecules containing thesequence -GCCCTT^(φ)xxxx-3′ (SEQ ID NO:12) at both 3′ ends (where xxxxis the complement of XXXX). Incubation of the PCR product withtopoisomerase results in cleavage at both termini and allows thecovalently activated PCR fragment to be ligated to vector DNA.

Regulatory elements required for expression include promoter or enhancersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. For example, a bacterial expression vectorincludes, but is not limited to, a promoter such as the lac promoter andfor transcription initiation the Shine-Dalgarno sequence and the startcodon AUG. Similarly, a eukaryotic expression vector includes, but isnot limited to, a heterologous or homologous promoter for RNA polymeraseII, a downstream polyadenylation signal, the start codon AUG, and atermination codon for detachment of the ribosome. Such vectors may beobtained commercially or assembled from the sequences described bymethods well-known in the art, for example the methods described abovefor constructing vectors in general.

In this invention transformation of the plasmid vector is into aprokaryotic host cell, such as a bacteria cell. In a preferredembodiment the host cell is E. coli.

Topoisomerase-based cloning has several advantages over conventionalligase-based cloning of PCR products. First, the topoisomerase procedurecircumvents any problems associated with addition of nontemplatednucleotides by DNA polymerase at the 3′ end of the amplified DNA. Anynontemplated base (N) at the 3′ end of a PCR product destined fortopoisomerase-based cloning (GCCCTT^(Ø)xxxxN-3′) (SEQ ID NO:13) willdissociate spontaneously upon covalent adduct formation, and willtherefore have no impact on the ligation to vector. Second, intopoisomerase-mediated cloning, the only molecule that can possibly beligated is the covalently activated insert and the insert can only betransferred to the vector. There is no potential for in vitro covalentclosure of the vector itself, which ensures low background. There isalso no opportunity for the inserts to ligate to each other (this can beguaranteed by using 5′-phosphate-terminated PCR primers), whichprecludes cloning of concatameric repeats. Third, there is no need toconsider the sequence of the DNA being amplified in designing the PCRprimers. It is commonplace in standard cloning to introduce arestriction site into the PCR primer and to cleave the PCR products withthat restriction enzyme to facilitate joining by ligase to vector. Incases where the sequence between the primers is not already known, itbecomes problematic to choose a site for the primer that is not presentin the amplified segment. This issue becomes even more relevant as PCRmethodology advances and very long targets (10-40 kbp) are amplifiedroutinely. The issue of internal topoisomerase cleavage sites (CCCTT(SEQ ID NO:17) or related pentapyrimidine elements) is not a significantimpediment to topoisomerase-based cloning. This is because thecleavage-religation equilibrium at internal sites strongly favors thenoncovalently bound state, and at those sites that are incised, only onestrand of the duplex is nicked. Internal cleavage sites can be inducedto religate by raising the salt concentration, which serves todissociate noncovalently bound topoisomerase and drive the reactionequilibrium to the left. In contrast, cleavage at sites near the 3′ endis virtually quantitative and is essentially irreversible until anacceptor DNA is provided.

Topoisomerase-based cloning strategies need not be limited to covalentactivation of the insert. By designing a plasmid polylinker such thatCCCTT (SEQ ID NO:17) sites are situated in inverted orientation oneither side of a restriction site, one can generate a linear vector withtopoisomerase sites at both 3′ ends. Once covalently activated bytopoisomerase, the vector “donor” can be used to clone any complementaryinsert “acceptor” (which must have 5′-OH termini), thereby precludingreligation of the vector without the insert. It is worth noting that thedonor complex formed upon cleavage by topoisomerase at a 3′ proximalsite is extremely stable. The donor molecule can be transferred nearlyquantitatively to a complementary acceptor even after many hours ofincubation of the covalent topo-DNA complex at room temperature. Indeed,the topo-linker complex can be denatured with 6 M guanidine HCl and thenrenatured spontaneously upon removal of guanidine with complete recoveryof strand transferase activity. Thus, a topoisomerase-activated vectorcan be prepared once in quantity and used as many times as needed formolecular cloning.

This invention provides a method of synthesizing polynucleotides. Oneembodiment comprises annealing a multiple number of duplex DNA strandsto form a branched substrate containing a sequence specifictopoisomerase cleavage site at each 3′ end; cleaving the branchedsubstrate by incubation with a sequence specific topoisomerase to form abranched topoisomerase complex; and incubating the branchedtopoisomerase complex with complementary monovalent and/or bivalent DNAacceptors. This method of polynucleotide synthesis is useful for invitro end-labelling, ligand tagging, molecular cloning.

In one embodiment the sequence specific topoisomerase is a vacciniatopoisomerase enzyme. In another embodiment the sequence specifictopoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT_(↓) (SEQ IDNO:16).

In one embodiment annealing of the duplex DNA strands is performed bymixing the DNA strands and heating to 65° C. for 5 minutes, and thenallowing the mixture to slow cool to room temperature. One skilled inthe art knows the procedures to follow for annealing duplex DNA.

In one embodiment three duplex DNA strands are used which form atrivalent Y-branched structure. Production of a Y-branched nucleic acidby the strand transfer reaction containing the trivalent linker can bedemonstrated by diagnostic restriction digestion of the reactionproducts. The yield of Y-branched products can be optimized byeliminating residual bivalent and monovalent linkers from the substratepreparation or by ensuring that all trivalent linkers were saturatedwith three bound topoisomerase molecules. Both conditions can be met, bygel-purifying the linker and by purifying the tri-covalently activatedspecies by sedimentation. As with bivalent ligation, the orientation ofthe Y-branched products can be controlled by manipulating the design ofthe linker, or by using asymmetric acceptors. Any head-to-head-to-headtype Y-branched product of trivalent strand transfer can, in theory, beorganized into a trivalent lattice by adding a second trivalent donorcomplex that is complementary to the “tail” of the original acceptorDNA. Donor substrates of higher order valence can be used to achievetopo-based synthesis of three dimensional lattices and polyhedra fromDNA. Topoisomerase-based synthesis offers a potentially powerfulalternative strategy for building complex biopolymers.

In one embodiment a duplex DNA strand is 5′ labeled and the 5′ labeledduplex DNA strand is annealed to the two duplex DNA strands to enableradiochemical purification of the substrate. Methods of radiochemicalpurification are known to those skilled in the art.

This invention provides a method of gene targeting. Gene targetinginvolves the introduction of DNA into a cell. The DNA is taken up intothe chromosomal DNA by virtue of a topoisomerase-bound donor duplex DNA.The bound topoisomerase seals the donor DNA to chromosomal DNA. Oneembodiment comprises cleaving a bivalent donor duplex DNA substratecontaining a sequence specific topoisomerase cleavage site by incubatingthe donor duplex DNA substrate with a sequence specific topoisomerase toform a topoisomerase-bound donor duplex DNA strand; and transfecting thetopoisomerase-bound donor duplex DNA to a suitable cell.

In one embodiment the sequence specific topoisomerase is a vacciniatopoisomerase enzyme. In another embodiment the sequence specifictopoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT_(↓) (SEQ IDNO:16).

Transfection may be performed by any of the standard methods known toone skilled in the art, including, but not limited to electroporation,calcium phosphate transfection or lipofection.

This invention provides a recombinant DNA molecule composed of segmentsof DNA which have been joined ex vivo or in vitro by the use of asequence specific topoisomerase and which has the capacity to transforma suitable host cell comprising a DNA sequence encoding polypeptideactivity.

In one embodiment the sequence specific topoisomerase is a vacciniatopoisomerase enzyme. In another embodiment the sequence specifictopoisomerase is a modified vaccinia topoisomerase enzyme. Inembodiments using vaccinia or modified vaccinia topoisomerase enzyme thecleavage site is an oligopyrimidine motif 5′ (C/T)CCTT_(↓) (SEQ IDNO:16).

This invention is further illustrated in the Experimental Detailssection which follows. This section is set forth to aid in anunderstanding of the invention but is not intended to, and should not beconstrued to, limit in any way the invention as set forth in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS I. Methods

A) Enzyme Purification:

Vaccinia DNA topoisomerase was expressed in Escherichia coli andpurified as described [9]. The heparin agarose enzyme fraction used inthe present study was the same preparation described previously [9]. Theenzyme was nearly homogeneous with respect to the 33 kDa topoisomerasepolypeptide, as determined by SDS-polyacrylamide gel electrophoresis.Protein concentration was determined using the Biorad dye reagent,taking bovine serum albumin as the standard.

B) Synthesis of 5′ Labeled Oligonucleotide Substrates:

Synthesis of DNA oligonucleotides via DMT-cyanoethyl phosphoramiditechemistry was performed by the Sloan-Kettering Microchemistry Laboratoryusing an Applied Biosystems model 380B or model 394 automated DNAsynthesizer according to protocols specified by the manufacturer.Oligonucleotides containing the CCCTT cleavage motif were labeled at the5′ end via enzymatic phosphorylation in the presence of [g³²P]ATP and T4polynucleotide kinase. Reaction mixtures (25 ml) typically contained 50mM Tris HCl (pH 8.0), 10 mM dithiothreitol, 10 mM MgCl₂, 0.1 mM ATP, 100mCi [g³²P]ATP, T4 polynucleotide kinase (20 units, Bethesda ResearchLaboratories), and 500 pmol of DNA oligonucleotide (DNA was quantitatedby A₂₆₀). Incubation was for 60 min at 37° C. Labeled DNA was freed ofprotein and radioactive nucleotide by electrophoresis through anon-denaturing 18% polyacrylamide gel. Full-sized labeledoligonucleotide was localized by autoradiographic exposure of the wetgel and the labeled DNA was recovered from an excised gel slice bysoaking the slice in 0.4 ml H₂O for 8 h at room temperature.Hybridization of labeled DNAs to complementary oligonucleotides wasperformed in 0.2 M NaCl by heating to 75° C. followed by slow cooling toroom temperature. Annealed substrates were stored at 4° C.

C) Topoisomerase-Based Cloning:

Reaction mixtures containing 50 mM Tris HCl (pH 7.5), 2 pmol oftopoisomerase, and either monovalent linker (0.6 pmol) or bivalentlinker (0.3 pmol) were incubated for 5 min at 37° C. A control reactioncontained topoisomerase but no DNA substrate. Each mixture was thensupplemented with 5′-OH HindIII-cut pUC18 DNA acceptor (380 fmol ofends) and incubated for another 5 min at room temperature. An aliquot (1ml) of each sample was used to transform E. coli DH5a using a BioRadGene Pulser electroporation apparatus. Preparation of Bacterial Cellsand Electrotransformation were carried out as prescribed by themanufacturer. Aliquots of transformed bacteria were plated on LB agarcontaining 0.1 mg/ml ampicillin.

II. Example 1 Sticky End Ligation

The vaccinia topoisomerase was capable of sticky-end ligation of duplexDNAs containing only 2 bases of potential complementarity, as shown inFIG. 1 (SEQ ID NOs:6 and 7). In this experiment, the “donor” was a24-mer hairpin oligonucleotide containing a single CCCTT motif (SEQ IDNO:17) (a “monovalent” substrate) with the scissile bond located 2 basesfrom the 3′ blunt end (FIG. 1A). The extent of cleavage of thissubstrate was proportional to enzyme concentration (FIG. 1A). Thetopoisomerase-DNA complex migrated as a discrete species during nativeagarose gel electrophoresis (FIG. 1C). Addition of unlabeled 5′hydroxyl-terminated CpG tailed linear pUC18 DNA (generated by digestionof pUC DNA with AccI followed by treatment with alkaline phosphatase)resulted in transfer of the topoisomerase-bound DNA strand to the linearDNA “acceptor.” The product of the strand transfer reaction was aradiolabeled 2.7 kbp linear form containing a hairpin end (FIG. 1C, lane2). AccI-restricted plasmid DNA containing a 5′-phosphate terminus wasinert as an acceptor (FIG. 1C, lane 3). [The requirement for a5′OH-terminated acceptor excluded the possibility that the reactionproducts might be formed by a conventional DNA ligase contaminating thetopoisomerase preparation]. Linear plasmid DNA containingnon-complementary 5′-OH overhangs generated by restriction with EcoRI(5′-AATT) or HindIII (5′-AGCT) were ineffective as acceptors (FIG. 1C,lanes 4 and 6), as was 5′-OH blunt-ended linear DNA generated byrestriction with SmaI (lane 5).

III. Example 2 Divalent Linkers as Donors

Two 46-mer DNA strands were annealed to form a “divalent” 46-bpsubstrate containing a topoisomerase cleavage site 4 nucleotides fromeach 3′ end (FIG. 2) (SEQ ID NOs:8 and 9). Successful annealing of theconstituent strands was evinced by the reduced mobility of the duplexmolecule during native gel electrophoresis (FIG. 3A, lane 2) compared tothat of the hairpin DNA (FIG. 3A, lane 1). Either the “flip” or “flop”monovalent hairpins were readily cleaved by vaccinia topoisomerase,resulting in the formation of a covalent protein-DNA adduct whichmigrated at 43 kDa during SDS-PAGE (FIG. 3B, lanes 2 and 4). Incubationof topoisomerase with the divalent duplex substrate yielded twocomplexes of 46 kDa and 72 kDa; the 46 kDa species represents a singlemolecule of topoisomerase bound covalently at one of the CCCTT (SEQ IDNO:17) cleavage sites; the 72 kDa complex arises by cleavage at bothsites on the same DNA molecule (FIG. 3B, lanes 6 and 8).

The monovalent hairpin DNA was transferred virtually quantitatively tolinear pUC DNA containing a complementary 5′-OH-AGCT overhang (FIGS.4A-4B, lane 2). Incubation of the bivalent topoisomerase-DNA complexwith the same acceptor yielded a complex set of products arising fromligation of the bivalent linker to two complementary ends of the linearpUC acceptor (FIGS. 4A-4B, lane 4). These included circular pUC andlinear pUC concatamers. A significant fraction of the pUC acceptormolecules were subject to bivalent end-joining, as reflected in thedistribution of EtBr-stained DNA products (FIG. 4A, lane 4). Allligation events were via the radiolabeled linker DNA, which becameincorporated into the reaction products (FIG. 4B, lane 4).

IV. Example 3 Molecular Cloning of DNA Using Vaccinia Topoisomerase

The ability of topoisomerase to join both ends of a linear DNA to acomplementary acceptor suggested an alternative approach to molecularcloning. In the scheme shown in FIG. 5, the “insert” was a bivalent46-bp linker containing CCCTT sites at both 3′ ends (SEQ ID NOs:10 and11). The sequence of the linker included restriction sites forendonucleases NdeI, BglII, and EcoRV. Cleavage of the bivalent linker bytopoisomerase generated a 4-base overhang complementary to a HindIIIrestriction site. The “vector” was pUC DNA that had been cleaved withHindIII and dephosphorylated with alkaline phosphatase. Addition of thevector to the bivalent topoisomerase-DNA donor complex should result incovalent joining of the insert to the vector. Upon transformation intoE. coli, those molecules that had been circularized should be able togive rise to ampicillin-resistant colonies. It was found that the yieldof ampicillin-resistant colonies from bacteria transformed with atopoisomerase reaction mixture containing linear pUC and the bivalentlinker was 110-fold higher than that observed for bacteria transformedwith control topoisomerase reactions containing linear pUC and eithermonovalent linker or no linker.

Plasmid DNA was recovered from cultures of six individual transformantsand analyzed by restriction endonuclease digestion in parallel withpUC18 plasmid DNA (FIG. 5B). [The restriction pattern for therecombinant clone pUC-T11 shown in FIG. 5C was indistinguishable fromthat of the five other clones, which are not shown]. Whereas thestarting pUC18 plasmid contains no sites for EcoRV and BglII, therecombinant clone contains a single site for each enzyme, attributableto the insertion of the bivalent linker, which contains theserestriction sites. Similarly, the starting plasmid contains a singleNdeI site, whereas the recombinant clone contains a second NdeI site inthe linker insert. The size of the novel NdeI fragment in pUC-T11indicated that the linker DNA was inserted within the pUC polylinker asexpected. This was confirmed by the finding that the recombinant plasmidhad lost the original HindIII site upon strand transfer by topoisomeraseto the HindIII overhang (the strand transfer reaction should generatethe sequences AAGCTA and TAGCTT at the plasmid-insert junctions, whichwould not be cut by HindIII). The restriction site for SphI, which islocated immediately next to the HindIII site in the polylinker, wasretained in all recombinant clones (not shown), indicating that loss ofthe HindIII site was not caused by deletions occurring during strandtransfer. Thus, the bivalent linker DNA was successfully cloned into thepUC18 vector in a simple procedure that—exclusive of the bacterialtransformation step—takes only 10 minutes to execute.

V. Example 4 Trivalent Linkers as Donors

Three 46-mer DNA strands were annealed to form a “trivalent” Y-branchedsubstrate containing a topoisomerase cleavage site 4 nucleotides fromeach 3′ end (FIG. 2). To optimize radiochemical purity of the substrate,one of the strands was 5′ radiolabeled and annealed to the two otherstrands, which were present in molar excess (FIG. 3A). The radiolabeledY-branched substrate migrated more slowly than a 46-bp linear duplexmolecule during native gel electrophoresis (FIG. 3A, lane 3). Anomalouselectrophoretic behavior of the Y molecule was also evident duringSDS-PAGE, where the trivalent substrate migrated at a positionequivalent to a 39 kDa protein (FIG. 3C, lane 1). The Y-branch structurewas cleaved quantitatively upon incubation with topoisomerase; threecomplexes were resolved, corresponding to Y-molecules with one, two, orthree covalent bound topo polypeptides (FIG. 3C). Most of the cleavedDNAs contained two or three bound topoisomerase molecules.

To test strand transfer by the trivalent donor complex, the Y-branchedmolecule was prepared by annealing equimolar amounts of the constituentstrands, each of which was radiolabeled. Although the three-strandY-form constituted the predominant product of the annealing reaction(FIG. 6A, lane 3), bivalent linkers were present as well (thesemolecules contain an unpaired “bubble” as indicated in FIG. 6). Theradiolabeled substrate was transferred quantitatively from thetopoisomerase-DNA donor complex to a linear pUC18 acceptor containing acomplementary 5′-OH-AGCT overhang (FIG. 6C, compare lanes 3 and 4). Acomplex array of multivalent ligation products was apparent byEtBr-staining and by autoradiography (FIGS. 6B-6C, lane 4). Theseincluded circular pUC and linear pUC concatamers as well as higher orderstructures (the species indicated by the bracket in FIG. 6C). None ofthe concatamers or higher order forms were observed in a control strandtransfer reaction containing a monovalent DNA linker (FIGS. 6B-6C, lane2).

VI. Example 5 Characterization of the Trivalent Strand Transfer Products

The recombinant molecules generated by topoisomerase-mediatedend-joining were analyzed further by digestion with restrictionendonucleases that cleave once within the pUC sequence. In FIG. 7, theanticipated products of bivalent end-joining by topoisomerase are shown,along with the restriction fragments expected for each product upondigestion with SspI and XmnI. The products of trivalent end-joining areillustrated in FIG. 8. Experimental results showing the spectrum ofstrand transfer products after digestion with SspI and XmnI are shown inFIG. 9. In this analysis, each linker, which upon cleavage generated atailed donor complex compatible with a HindIII restriction site, wastested with two acceptor molecules, one bivalent and one monovalent. Thebivalent acceptor was linear pUC19 containing 5′-OH HindIII overhangs onboth ends. Strand transfer of a polyvalent linker to the bivalentacceptor allows for the formation of circular and linear concatamers ina head-to-head, tail-to-tail, or head-to tail fashion, as shown in FIG.7. The monovalent acceptor was pUC19 containing a 5′-OH HindIII site atone end and a 5′-phosphate AccI site at the other end. Transfer of thelinker by topoisomerase to the AccI terminus is precluded completely ontwo grounds; first, because the ends are not complementary and second,because topoisomerase cannot religate to a 5′-phosphate strand. Amonovalent acceptor will react with the topoisomerase donor complex atavailable compatible termini, but will not be able to form circles orconcatameric arrays. The structures of the various species can thus beinferred by direct comparison of the restriction digests from reactionin which monovalent, bivalent, and trivalent linkers were reacted withmonovalent and bivalent acceptors.

Consider the SspI digests of topoisomerase strand transfer products inFIG. 9A. The monovalent linker was joined to either end of the bivalentpUC19 acceptor, but could not support circularization or dimerization.Hence the products were cleaved by SspI to yield two fragments derivedfrom linear monomers (FIG. 9A, lane 1) (see FIG. 7). Ligation of thebivalent linker to bivalent acceptor yielded three additional products,a 4.1 kbp fragment diagnostic of head-to-head multimer formation, a 1.3kbp fragment indicative of tail-to-tail ligation, and a 2.7 kbp speciesthat derived from a circular molecule (FIG. 9A, lane 3). Ligation of thebivalent linker to a monovalent acceptor yielded the 4.1 kbphead-to-head fragment, but no fragments indicative of tail-to-tail orcircular products (FIG. 9A, compare lanes 3 and 4). This was preciselyas expected, because the AccI “tail” was inert for strand transfer.Reactions containing the trivalent Y-linker and bivalent acceptoryielded two novel high molecular weight products not observed for thebivalent linker (FIG. 9A, lane 5). The largest product (indicated by thearrowhead in FIG. 9A), which was also observed with trivalent linker andmonovalent acceptor (FIG. 9A, lane 6), must correspond to a Y-branchedrecombinant containing three pUC molecules ligated in head-to headfashion. The length of each arm is predicted to be 2 kbp. Theelectrophoretic mobility of this species was anomalously slow, asexpected for a branched DNA. The higher order complex unique to thebivalent acceptor was presumed to be a Y-branched product containingpUC19 DNA ligated in a mixed head-head and head-tail orientation.

Digestion of the strand transfer products with XmnI confirmed andextended these findings (FIGS. 9B-9C). The digest of a reactioncontaining labeled bivalent linker and unlabeled bivalent pUC acceptoryielded diagnostic linear fragments of 3.7 kbp (head-to-head multimer),1.7 kbp (tail-to-tail multimer) and 2.7 kbp (circle). These productswere detected by EtBr-staining and by autoradiography (FIG. 9B, lanes1). The 1.7 kbp species indicative of tail-to-tail ligation migratedjust ahead of a 1.85 fragment (derived either from end-tagged linearmonomers or from head-to-tail multimers). The 1.7 kbp species was absentfrom the digest of products formed with the monovalent pUC acceptor(FIG. 9B, lanes 2). Similarly, the 2.7 kbp species and the radiolabeled0.8 kbp fragment (diagnostic of ligation to the “tail” end of pUC) wereabsent from the monovalent acceptor digest (FIG. 9B, lane 2).

The XmnI digest of products formed with labeled trivalent linker andbivalent pUC19 acceptor contained four unique species not seen with thebivalent linker (FIG. 9B, compare lanes 3 and 1). Three of thesemolecules were readily apparent as high molecular weight EtBr-stainedbands. The fourth species migrated barely in advance of the head-to-headlinear fragment and was best appreciated in the autoradiograph (FIG. 9C,lane 3). These molecules correspond to the four possible Y-branchstructures shown in FIG. 8. A priori, if there was no bias in ligationorientation, one would expect a 1:3:3:1 distribution of head-head-head,head-head-tail, head-tail-tail, and tail-tail-tail isomers. Indeed, thisis what was observed experimentally (FIG. 9B, lane 3). Consistent withthe predicted structures of the Y-branched products, only the largestspecies (head-head-head) was detected in the reaction of trivalentlinker with monovalent pUC acceptor.

References:

-   1. Chen, J., and Seeman, N. C. (1991) Nature 350: 631-633.-   2. Cheng, S., et al. (1994) Proc. Natl. Acad. Sci. USA 91:    5695-5699.-   3. Clark, J. M. (1988) Nucleic Acids Res. 16: 9677-9686.-   4. Morham, S. G., and Shuman, S. (1992) J. Biol. Chem. 267:    15984-15992.-   5. Shuman, S. (1991a) J. Biol. Chem. 266: 1796-1803.-   6. Shuman, S. (1991b) J. Biol. Chem. 266: 11372-11379.-   7. Shuman, S. (1992a) J. Biol. Chem. 267: 8620-8627.-   8. Shuman, S. (1992b) J. Biol. Chem. 267: 16755-16758.-   9. Shuman, S., et al. (1988) J. Biol. Chem. 263: 16401-16407.-   10. Shuman, S., et al. (1989) Proc. Natl. Acad. Sci. USA 86:    9793-9797.-   11. Shuman, S., and Moss, B. (1987) Proc. Natl. Acad. Sci. USA 84:    7478-7482.-   12. Shuman, S., and Prescott, J. (1990) J. Biol. Chem. 265:    17826-17836.-   13. Stivers, J. T., et al. (1994) Biochemistry 33: 327-339.

What is claimed is:
 1. A method of producing a ligated DNA moleculecomprising: contacting an acceptor duplex DNA molecule with a donorduplex DNA molecule that comprises: a sequence specific type Itopoisomerase bound on at least one end of the molecule and at least oneof an origin of replication and a selection marker, under conditions inwhich the sequence-specific type I topoisomerase ligates the donorduplex DNA molecule to the acceptor duplex DNA molecule, to produce aligated DNA molecule.
 2. The method of claim 1, wherein the type Itopoisomerase is a vaccinia type I topoisomerase.
 3. The method of claim1, wherein the acceptor duplex DNA molecule has a 5′ terminal hydroxylon each end.
 4. The method of claim 1, further comprising introducingthe ligated DNA molecule into a cell.
 5. The method of claim 4, whereinthe cell is an Escherichia coli cell.
 6. The method of claim 1, whereinthe donor duplex DNA molecule comprises an origin of replication.
 7. Themethod of claim 1, wherein the donor duplex DNA molecule comprises aselection marker.
 8. The method of claim 1, wherein the acceptor duplexDNA molecule is generated by a polymerase chain reaction (PCR).
 9. Themethod of claim 1, wherein the donor duplex DNA molecule is linear.